The TGF-.beta. superfamily is one of the largest groups of polypeptide growth and differentiation factors. A variety of structural and functional criteria have been used to group the superfamily into three classes: (1) TGF-.beta.s; (2) activins; (3) bone morphogenetic proteins (BMPs). Members of these groups mediate a wide range of biological processes in vertebrates and invertebrates, includings regulation of cell proliferation, differentiation, recognition, and death, and thus play a major role in developmental processes, tissue recycling, and repair (J. Wrana and L. Attisano, "Mad-related Proteins in TGF-.beta. Signaling," TIG 12:493-496, 1996).
Genetic and biochemical studies indicate that TGF-.beta. and related factors, including activin, BMPs and their Drosophila counterpart, Decapentaplegic (DPP) signal to their target cells by simultaneously contacting two transmembrane receptor serine/threonine kinases, known as the type I and type II receptors (J. Massague, "TGF.beta. Signaling: Receptors, Transducers, and Mad Proteins," Cell 85:947-950, 1996). Receptor activation occurs upon binding of ligand to the type II receptor, which then recruits and phosphorylates the type 1 receptor in its glycine and serine-rich domain. Once phosphorylated, receptor I is activated and then propogates the signal to downstream targets (L. Atisano et al., "Activation of the signaling by the activin receptor complex." Mol. Cell. Biol. 16:, 1066-1073). Also, constitutively active type I receptors (L. Atisano et al., "Activation of the signalling by the activin receptor complex." Mol. Cell. Biol. 16: 1066-1073; V. Wiersdorff et al., "Mad Acts Downstream Of Dpp Receptors, Revealing A Differential Requirement For Dpp Signaling In Initiation and Propagation Of Morphogenesis in the Drosophila Eye," Development 122:2153-2162, 1996), appear to signal biological responses in the absence of ligand and receptor II (RII) (R. Weiser et al, "GS domain mutations that constitutively activate TGF.beta. R1, the downstream signaling component in the TGF.beta. receptor complex," EMBO J., 14:2199-2208), which indicates the role of the type I receptor (RI) as the downstream or the essential transducing element.
Although, the molecular workings of the TGF-.beta. receptors are reasonably well understood, little is known of the downstream intermediate targets that transmits the Ser/Thr kinase receptor signals from the cell membrane to the nucleus. Recent studies indicate that signaling by TGF-.beta.-like molecules may be transduced by a set of evolutionary conserved proteins known as Smads, which upon activation directly translocate to the nucleus, where they may activate transcription (F. Liu et al., "A Human Mad Protein Acting As A BMP-regulated Transcriptional Activator," Nature 381:622-623, 1996). Five Smad proteins, designated Smad 1-5, have been so far characterized in vertebrates (R. Derynck and Y. Zhang, "Intracellular Signalling: The Mad Way To Do It," Curr. Biol. 6:1226-1229, 1996). These factors are related to the intracellular mediators of DPP signaling, mothers against dpp (MAD) in the fruitfly Drosophila melanogaster (L. Raftery et al., "Genetic Screens to identify elements of the Decapentaplegic Pathway In Drosophila," Genetics 139:241-254 (1995)., and to the Sma genes from the nematode Caenorhabditis elegans (C. Savage et al., "C. Elegans Genes Sma-2, Sma-3 and Sma-4 Genes Define A Conserved Family Of TGF-.beta. Pathway Components," Proc. Natl. Acad. Sci. USA 93:790-794, 1996; R. Derynck and Y. Zhang, "Intracellular Signalling: The Mad Way To Do It," Curr. Biol. 6:1226-1229, 1996). Signaling causes Smads to form hetero-oligomers. BMP signaling stimulates Smad1 to form a complex with Smad4; activin signaling stimulates Smad2 to form a complex with Smad4 (G. Lagna et al., "Partnership Between DPC4 and Smad Proteins in TGF-.beta. Signaling Pathways," Nature 383:832-836, 1996). It is speculated that common responses to multiple ligands might be mediated by overlap in the regulation of some Smad proteins, while localized expression and activation of specific Smads may mediate specific biological responses (J. Wrana and L. Attisano, "MAD-related Proteins in TGF-.beta. Signalling," TIG 12:493-496, 1996).
There is little information at present as to how these Smads and homologous proteins might elicit cellular responses. Analysis of Smad1 suggests that this protein resides predominantly in the cytoplasm in unstimulated cells, but accumulates in the nucleus upon activation of BMP signaling pathways (P. A. Hoodless et al., "MADR1, A MAD-related Protein That Functions In BMP2 Signaling Pathways," Cell 85:489-500, 1996). Similarly, the nuclear localization of a lacZ-Smad2 fusion protein, expressed in Xenopus ectoderm explants, is enhanced by the addition of activin (J. C.Baker & R.Harland, Genes Dev. 10:1880-1889, 1996). Also, a lacZ fusion with a C-terminal fragment of Smad2 displays constitutive nuclear localization, suggesting that the N-terminal domain can act to retain Smad proteins in the cytoplasm (J. C.Baker & R.Harland, Genes Dev. 10:1880-1889, 1996). Although the nature of the signals that control Smad protein localization is not clear, the function of these intracellular proteins is to transmit signals from the cytoplasm to the nucleus resulting in the activation of target gene expression.
Regardless of the precise nature of the pathway, there is evidence that TGF-.beta. plays a role in the pathogenesis of impaired wound healing, human fibrotic diseases, autoimmune diseases, atherosclerosis and malignancy. Over-production or under-production of TGF-.beta.s underlie these pathologies. Importantly, manipulation of TGF-.beta. levels has been shown to reverse these pathologies in experimental models of human disease. Increased TGF-.beta. normalizes the wound healing defects in aged or glucocorticoid treated rats. Administration of TGF-.beta. has been shown to ameliorate EAE, a model of multiple sclerosis, and experimental arthritis. Antibodies to TGF-.beta. exacerbate the symptoms in these models.
TGF-.beta. has at least two important roles in cancer. It is growth inhibitory to many cells so that loss of responsiveness to TGF-.beta. through mutation of receptor or Smad proteins results in uncontrolled proliferation. Second, it is highly immunosuppressive so that tumor cells no longer responsive to TGF-.beta. themselves, up-regulate the expression of TGF-.beta. to protect themselves from the immune system. Increased expression of TGF-.beta. may also enhance the ability of the tumor cells to migrate to new sites during metastasis.
Breast Cancer: TGF-.beta. mRNA has been quantitated in human breast carcinomas. In a group of 24 samples, overexpression of TGF-.beta. was detected in 75% of the tumors. The increase did not correlate with grade, estrogen responsiveness, progesterone receptor status or lymph node involvement [Christeli et al. (1996) Oncol. Rpts. 3(6):1115-1118].
Tissue factor (TF), the cellular initiator of the protease blood coagulation cascade, is expressed in the stroma of breast tumors progressing to invasive cancer. This expression is induced by conditioned media (CM) from breast cancer cells and the CM effect is blocked by anti-TGF-.beta. antibodies. Tumor cell-derived TGF-.beta. induction of stromal cell TF is an early event in progression to invasive breast cancer [Vrana et al. (1996) Cancer Res. 56(21):5063-5070].
The antiproliferative effects of calcitriol and lexicalcitol (KH1060) on human breast epithelial cells has been shown to be blocked by the presence of anti-TGF-.beta. [Mercier et al. (1996) Biochem. Pharmacol. 52(3):505-510].
The antiproliferative effects of the antiprogestin onapristone correlated with its ability to increase TGF-.beta. production of human breast cancer cell lines [Dannecker et al. (1996) Ann. of Onco. 7(4):391-395].
81% of breast cancer patients had elevated plasma TGF-.beta. levels of more than two standard deviations above the normal mean [Kong (1995) Ann. Surg. 222:2].
Colon and Gastric Cancer: Increased TGF-.beta. expression in colorectal tumors has been shown to be correlated with poor prognosis [Robson et al. (1996) Brit. J. Cancer. 74(5):753-758]. In addition, plasma TGF-.beta. levels are higher in patients with colorectal cancer. Levels of mRNA and protein are higher in the tumor tissue [Tsushima et al. (1996) Gastroenterol. 110(2):375-382].
Alteration in type II receptor (RII) has been found in 57% of adenomas and 85% of colon tumors in 10 patients studied, indicating a strong association between RII mutations and progression of disease [Akiyama (1997) Gastroenterol. 112(1):33-39]. Evaluation of 138 human tumors for type II receptor mutations concluded that these mutations were associated with a subset of ulcerative colitis carcinomas, common (81%) in sporadic colorectal cancers and rare in gastric and esophageal cancer [Souza et al. (1997) Gastroenterol. 112(1):40-45].
In addition, examination of RII mutations in 112 cases of gastrointestinal and hepatobiliary cancer found replication error in 17 tumors and also RII mutations in 10 of those 17 tumors (3 of 4 gastric and 7 of 10 colorectal) [Togo et al. (1996) Cancer Res. 56(24):5620-5623].
Glioma: Gliomas express immunosuppressive TGF-.beta.. In a rat model, 9L gliomasarcoma cells were modified to express a TGF-.beta. antisense plasmid. 11 of 11 animals survived for 12 weeks compared to 2 of 15 control animals with 9L cells. In animals treated with TGF-.beta. antisense therapy, there was no evidence of residual tumor. Inhibition of TGF-.beta. expression significantly enhanced tumor-cell immunogenicity [Fakhrai et al. (1996) Proc. of the Natl. Acad. of Sci. USA 93(7):2909-2914]. However, at present, human malignant gliomas are resistant to all current therapeutic approaches.(See review: Weller and Fontana "Malignant glioma cells". Brain Res. Rev. 21(2):128-151, 1995).
Lung Cancer: The association of TGF-.beta. has also been made with lung cancer. In one study, elevated TGF-.beta. levels (.about.3 fold) were detected in the plasma of 50% (27/54) lung cancer patients [Kong et al. ((196) Lung Cancer 16(1):47-59].
Prostate Cancer: Increased levels of urinary TGF-.beta. 1 and plasma TGF-.beta. 2 have also been reported in patients with prostate cancer [Perry et al (1997) Urology 49(1):151-155]. Review: [Steiner (1995) World J. Urol. 13(6):329-336]. Fenretinide (4-HPR), a retinoid derivative with anti-neoplastic activity, when used to treate prostate cancer cells (PC-3) caused apoptosis, increased TGF-.beta. 1 mRNA and protein levels and increased TGF-.beta. receptor 1 expression. The cytotoxixity was blocked by addition of anti-TGF-.beta. 1. A resistant breast cell line BT-20 did not show increased TGF-.beta. in response to 4-HPR [Roberson et al. (1997) Cell. Growth and Diff. 8(1):101-111].
Renal Cell carcinoma: Levels of latent TGF-.beta. 1 produced in renal cell carcinoma patients has been reported to be approximately 10-fold higher than in healthy controls, which may be responsible for the local immunosuppressive effects seen within the tumor [Junker et al (1996) Cytokine 8(10):794-798].
Other Cancers: The above-specified cancers are by no means the only types having an association with TGF-.beta.. They are simply illustrative. These examples make it evident that a better understanding of the role and function of members of the TGF-.beta. cascade is needed in order to circumvent the onset and progression of many types of cancer.